We continue to investigate the role of membrane CPE and secretogranin III as sorting receptors for targeting POMC to the regulated secretory pathway (RSP). CPE knockout (KO) mice show defective trafficking of POMC in primary cultures of anterior pituitary cells since 50% of the newly synthesized POMC was targeted for degradation in the absence of CPE. However, some of the remaining POMC was sorted into the RSP, presumably by Secretogranin III (SgIII). SgIII is involved in trafficking of chromogranin A (CgA) to the RSP. Using RNA interference to knock down SgIII and CPE, we showed that both proteins affect the normal secretion of POMC in AtT20 cells, i.e. POMC was secreted at an elevated rate through the constitutive secretory pathway when either CPE or SgIII are reduced. When both are knocked-down, the affect is augmented, suggesting that POMC trafficking is dependent on both proteins for efficient trafficking to the RSP. In collaboration with Dr. Josh Park, Uni. of Toledo, we showed that snapin connects a microtubule motor complex consisting of kinesin-2, cytoplasmic dynein, and dynactin to the cytoplasmic tail of CPE on POMC vesicles to mediate their transport in anterior pituitary AtT-20 cells. Snapin directly binds to CPE cytoplasmic tail and interacts with microtubule motors. Overexpression of snapin reduced process-localization, processivity and velocity of movement of ACTH/POMC vesicles, similar to overexpression of CPE C-terminal tail. The intracellular interaction between snapin and dynactin was confirmed using quantum dot conjugated snapin. Knockdown of snapin decreased stimulated ACTH secretion. Moreover, A kinase anchor protein 150 (AKAP150), a scaffold for protein kinase A and calcineurin, associate with snapin-microtubule motor complex to facilitate the process-localization of ACTH/POMC vesicles. Thus, our study uncovered a new molecular complex that mediates and possibly regulates post-Golgi transport of ACTH/POMC vesicles to the process terminals of AtT20 cells. In collaboration with Dr. Bruno Tota (Uni. of Calabria), pGlu-serpinin, a CgA-derived peptide was found to have positive inotropic activity in cardiac function, with no change in blood pressure and heart rate. pGlu-serpinin acts through a &#61538;1-adrenergic receptor/adenylate cyclase/cAMP/PKA pathway and emerges as a novel-adrenergic inotropic and lusitropic modulator of the myocardium in response to sympathochromaffin stimulation. We have also investigated the effect of pGlu-serpinin on cardioprotection. Using normotensive (WKY) and hypertensive (SHR) rats as models; we showed that pGlu-serpinin mimicked pre-conditioning and post-conditioning-induced cardioprotection. In both WKY and SHR rats, pGlu-serpinin improved left ventricle function recovery after ischemia. Moreover, it reduced ischemicinduced contracture state and decreased infarct size. In pGlu-serpinin mediated post-conditioning pharmacological cardiac protection, the mechanism involved the activation of the reperfusion injury salvage kinase (RISK) pathway. CPE-KO mice have defects in their nervous system function, including learning and memory. This defect is due to the loss of neurons in the CA3 region of the hippocampus due to the stress of ear tagging and tail clipping for genotyping and the emotional stress of maternal separation during weaning (3 weeks). The neurodegeneration was prevented by the drug carbamazepine, an anti-epileptic drug, demonstrating that the stress associated with weaning induced epileptic-like seizures resulting in neuronal cell death in the absence of CPE. We examined the effect of restraint stress on CPE expression in hippocampal neurons. When mice were subjected to mild chronic stress (1h/d for 7d), which increases glucocorticoid secretion, the mice showed an increase in CPE mRNA and protein in the hippocampus, and no neuronal degeneration was evident. When hippocampal neurons were treated with synthetic glucocorticoid, dexamethasone, there was a significant increase in CPE mRNA and protein in the cells. These observations suggest that the increase in CPE may mediate neuronal survival during stress and lack of CPE results in neuronal degeneration. We then applied recombinant CPE to rat hippocampal neurons in culture and showed increased survival and neuroprotective effect of CPE on these neurons when subjected to oxidative stress with hydrogen peroxide treatment or glutamate cytotoxicity. Since CPE can act as a neuroprotective molecule with trophic properties, we have recently named it neurotrophic factor-alpha1 (NF-alpha1) to indicate this when CPE functions as a trophic factor. Recently we have investigated the role of NF-alpha1 in preventing restraint stress-induced depression. Prolonged (6h/d for 21 days), but not short-term (1h/d for 7d) restraint stress reduced fibroblast growth factor 2 (FGF2) in the hippocampus, leading to depressive-like behavior in mice. We found that mice after short-term restraint stress increased hippocampal NF-alpha1, FGF2 and doublecortin, a marker for immature neurons, suggesting increased neurogenesis. Indeed we showed that in cultured hippocampal neurons, exogenous NF-alpha1 could increase FGF2 expression. After prolonged restraint stress, mice showed decreased NF-alpha1 and FGF2 levels. Moreover, NF-alpha1-KO mice exhibited severely reduced hippocampal FGF2 levels and immature neuron numbers in the subgranular zone. These mice displayed depressive-like behavior that was rescued by FGF2 administration. Thus, NF-alpha1 prevents stress-induced depression by up-regulating hippocampal FGF2 expression which leads to enhanced neurogenesis and anti-depressant activity. The expression of NF-alpha1 and a splice variant isoform, CPE_deltaN was examined in mouse embryos to determine if it could play a role in development and neuroprotection of embryos. We found that WT CPE and CPE-deltaN mRNA was expressed as early as day E5.5 and increased each day, peaking at E8.5, falling slightly at E9.5 prior to expression of the endocrine system. CPE mRNA expression decreased sharply at E 10.5-11.5 to below E5.5 levels and then increased sharply at E12.5 in parallel with the development of the endocrine system and continued to increase to adulthood. However, CPE-deltaN mRNA increased maximally at E10.5 followed by a decrease at E11.5-12.5, and then a small increase till PN1. In contrast to NF-alpha1, CPE-deltaN is virtually absent in the adult brain. In situ hybridization studies indicate that WT CPE and CPEdeltaN mRNA are expressed primarily in the fore brain and somites in mouse embryos. We showed that CPE-deltaN plays a role in protecting embryonic cortical and hippocampal neurons from glutamate neurotoxicity and oxidative stress through up-regulating FGF2 expression which then increased the pro-survival protein, BCl2. We also studied the role of NF-alpha1 during embryonic development of the nervous system using neurospheres to study proliferation and differentiation. Exogenous addition of NF-alpha1 to E13.5 neocortex-derived neurospheres, which contains stem cells and neuroprogenitors, resulted in reduced proliferation of the neurospheres without causing cell death. NF-alpha1 down-regulated the wnt-pathway in the neurospheres leading to reduced levels of beta-catenin which is known to enhance proliferation. Differentiation studies using neurospheres from 7d cultures that were dissociated into single cells and cultured for an additional 5d showed an increase in astrocytes in the presence of NF-alpha1, without altering the percentage of neuronal and oligodendrocyte populations. Interestingly, dissociated cells from neurospheres derived from NF-alpha1-KO mouse embryos showed decreased astrocytes and increased neurons. Our results suggest a novel role of NF-alpha1 as an extracellular signal to differentiate neural stem cells into astrocytes.